Genetically modified plants and plant cells comprising heterologous heavy metal transport and complexation proteins

ABSTRACT

The present invention relates to genetically modified plants and plant cells, comprising nucleotide sequences encoding heterologous heavy metal transport protein.

FIELD OF THE INVENTION

[0001] The present invention is in the field of genetically modified plants and plants cells having improved heavy metal tolerance and accumulation due to increased plant growth and biomass production based upon the expression of exo-cytoplasmic heavy metal resistance system (efflux and complexation).

[0002] More particularly, the present invention is related to genetically modified plants and plant cells, comprising nucleotide sequences encoding heterologous heavy metal transport proteins and exocytoplasmic metal binding proteins of various origins.

BACKGROUND OF THE INVENTION AND STATE OF THE ART

[0003] Heterologous nucleic acid sequences, coding for heavy metal resistance, were functionally expressed in plants, to improve their tolerance against these toxic elements. The heterologous heavy metal resistance genes, in casu represent either heavy metal efflux systems or functions involved in heavy metal sequestration.

[0004] Until present, only cytoplasmic functions that provide increased heavy metal resistance were expressed in plants.

[0005] 1. Expression of Heterologous Metallothionein and Phytochelatines in Plants

[0006] Metallothioneins and phytochelatines, which are rich in cystein sulfhydryl residues that bind and sequester heavy metal ions in very stable complexes (Karin, 1985), are found in eukaryotic organisms, but recently also in Synechococcus. Various MT genes—mouse MTI, human MTIA (alpha domain), human MTII, Chinese hamster MTII, yeast CUP1, pea PsMTA—have been transferred to tobacco, cauliflower or Arabidopsis thaliana (Lefebre et al., 1987; Maiti et al., 1988, 1989, 1991; Misra and Gedamu, 1989; Evans et al., 1992; Yeargan et al., 1992; Brandle et al., 1993; Pan et al., 1993; Elmayan and Tepfer, 1994; Hattori et al., 1994; Pan et al., 1994a, b; Hasegawa et al., 1997). As a result, varying degrees of enhanced Cd tolerance have been achieved, being maximally 20-fold compared with the control. Metal uptake levels were not dramatically changed; in some cases there were no differences, in others maximally 70% less or 60% more Cd was taken up by the shoots or leaves. Only one study has been reported on a transgenic plant generated with MT of plant origin. When pea (Pisum sativum) MT-like gene PsMTA was expressed in Arabidopsis thaliana, more Cu (several-fold in some plants) accumulated in transformed than in control plants (Evans et al., 1992).

[0007] 2. Heterologous Expression of Heavy Metal Reduction

[0008] The only example known is the mer operon of Tn21 of Shigella flexneri, whose expression in plants results in the reduction mercury (Hg²⁺) in its metallic form (Hg⁰). This metallic mercury is volatilized out of the cell (Rugh et al. 1996).

AIMS OF THE INVENTION

[0009] The present invention aims to provide a new way in obtaining plants and plant cells with improved heavy metal tolerance characteristics, and possibly heavy metal accumulation.

[0010] Another aim of the present invention is to provide such plants and plant cells which allow increased heavy metal resistance for revegetation and phytostabilisation of heavy metal contaminated sites.

[0011] A further aim of the present invention is to provide plants and plant cells, characterised by increased heavy metal accumulation combined with increased heavy metal tolerance which allow phytoextraction of heavy metals (inclusive rhizofiltration).

[0012] A last aim of the present invention is to provide a method which results in the possibility to improve important agriculture crop species with high biomass production in their heavy metal tolerance and accumulation.

SUMMARY OF THE INVENTION

[0013] The present invention is related to genetically modified plant and plant cell having improved (induced or increased) heavy metal resistance, comprising at least one nucleotide sequence encoding one or more heterologous heavy metal transport and/or sequestration proteins of various prokaryotic or eukaryotic origins.

[0014] Said transporters are preferably membrane proteins, which result in reduced toxicity due to the efflux of heavy metals from the cells and being preferably selected from the group consisting of P-type ATPases, 3 component efflux pumps, ABC transporters and CDF proteins (Cation Diffusion Facilitator proteins).

[0015] The family of the P-type ATPases is preferred, because of their advantage that for functional resistance only one protein is required.

[0016] Said proteins are found in both prokaryotic and eukaryotic organisms including plants.

[0017] Another advantage of said transporters is found as resistance mechanisms against many toxic trace elements of environmental concern, such as copper, cadmium, lead, zinc and silver.

[0018] Unexpectedly, it was not necessary to make structural changes in the coding sequence of said proteins, like it is necessary for the merA gene in order to obtain functional expression in plants (Rugh et al., 1996).

[0019] Preferably, the gene incorporated in the plants or plant cells is a gene encoding a bacterial P-type ATPase, preferably the cadmium ATPase, such as the cadA gene.

[0020] Another aspect of the present invention is related to a method for inducing (or improving) increased heavy metal resistance into a plant or a plant cell, said method comprising the following steps:

[0021] preparing at least one nucleotide sequence encoding one or more heterologous heavy metal transport and/or sequestration proteins, operably linked to one or more regulatory sequences active into a plant,

[0022] transforming a plant or plant cell with said nucleotide sequence and,

[0023] possibly regenerating a (transgenic) plant from the transformed plant or plant cell.

[0024] According to a second embodiment of the present invention, the system is based upon a prokaryotic heavy metal sequestration system, such as the pcoA family protein (more preferably the pcoA gene).

[0025] The various nucleotide sequences encoding heterologous heavy metal transport proteins can be deleted partially from non-specific nucleotide sequences which are not involved in efficient heavy metal transport or accumulation.

[0026] Said genetic sequences could be incorporated in a vector for the transfection of said plants or plant cells, such as the pBI121 vector, as described in the FIG. 1, said vector being advantageously an E. coli/Agrobacterium/plant shuttle vector, said vector comprising preferably a CaMV 35S promoter (a strong promoter constitutively expressed in plants).

[0027] Preferably, the system was introduced in the plants, such system allowing the transformation of plants with the Agrobacterium tumefaciens technology.

[0028] The present invention can be used for phytoremediation of contaminated sites, or for the preparation of a medicament or food/feed supplements containing trace elements.

[0029] The plant or plant cell according to the invention has various applications, especially in the field of phytoremediation of contaminated sites with heavy metal (such as areas having grounds contaminated with heavy metals or aquatic or semi-aquatic areas contaminated with heavy metals). The phytoremediation of contaminated sites comprises usually the step of revegetation, phytostabilisation, phytoextraction of soil and/or water contaminated with trace elements of said heavy metal.

[0030] Another application of the plant or plant cell according to the invention is a medical and agro-industrial application, wherein said plant or plant cell are natural sources for trace elements.

[0031] Therefore, a last aspect of the present invention is related to a pharmaceutical composition of food or feed compositions or additives, containing said plant or plant cells with heavy metal trace elements.

SHORT DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 is a schematic representation of the cloning of cadA in pBI121.

[0033]FIG. 2 is a leaf disk-test with Nt WT SR1 (wild type), Nt PBI14 (pBI121) and Nt Cd 309 (pBI121-cadA) on 350 μM Cd and control medium without Cd.

[0034]FIG. 3 represents the regeneration and growth of Nt WT SR1 (wild type), Nt PBI14 (pBI121) and Nt Cu122 (pBI121-pcoA) on 100 μM Cu, the plant growth being shown from above (left) and top (right).

DETAILED DESCRIPTION OF THE INVENTION

[0035] Heterologous Expression of cadA

[0036] The heavy metal efflux system was cadA, a member of the P-type heavy metal efflux ATPase family of proteins found both in prokaryotic and eukaryotic organisms. P-type ATPases are all cation pumps, either for uptake, for efflux or for cation exchange. These enzymes have a conserved aspartate residue that is transiently phosphorylated from ATP during the transport cycle, hence the name ‘P-type’ ATPase (Silver et al., 1993).

[0037] The cadA gene from Staphylococcus aureus was amplified by PCR and cloned in the pBI121 vector.

[0038] During PCR, appropriate plant specific translation signals were added as well as XbaI and BamHI restriction sites, allowing cloning of the insert in the correct orientation.

[0039] The cadA fragment was cloned in the Escherichia coli/Agrobacterium/plant shuttle vector pBI121. In this vector, cadA expression is derived from the CaMV35S promotor, a strong promoter constitutively expressed in plants. The system was introduced in the plant Nicotiana tabacum cv. Petit Havana line SR1 via an Agrobacterium tumefaciens transformation (Horsch et al., 1985). The selection marker used was kanamycine.

[0040] Kanamycine resistant transformants were obtained after transformation. All the kanamycine resistant transformants tested showed an increased resistance to cadmium (tested by a leaf disk assay) compared to the wild type and transformant with the pBI121 vector without gene (FIG. 1). This proves that the CadA P-type ATPase can be functionally expressed in plants, resulting in an increased resistance of the plant to the trace element (in casu cadmium).

[0041] It can be expected that for other members of the P-type ATPase family, which form a family of closely related proteins (both structural and functional) the same positive effect on resistance to specific trace elements will be found. Until present, P-type ATPases from both prokaryotic and eukaryotic have been identified that were found to interact with Zn, Cd, Pb, Cu and Ag (see table 1). It can not be excluded that P-type ATPases, encoding resistance to other trace elements including radioisotopes, will be identified. TABLE 1 different representatives of the family of P-type ATPases, from prokaryotic and eukariotic origin, which encode resistance against trace elements such as Zn, Cd, Pb, Cu and Ag. Sequence Gene ID Metals Reference CadA P20021 Cd, Zn and Nucifora et al. 1989 Pb Rensing et al. 1998 ZntA P37617 Zn and Pb Rensing et al. 1997 Rensing et al. 1998 CopF Non Cu van der Lelie and available Borremans unpublished PbrA Not Pb Borremans et al, 2000 available SilP AF067954, Ag Gupta et al, 1999 nucleotide sequence sil operon Menkes' Q04656 Cu Vulpe et al. 1993 disease Wilsons' 1J08344 Cu Pethrukin et al. 1993 disease

[0042] Heterologous Expression of pcoA

[0043] The other heavy metal resistance system is involved in exo-cytoplasmic heavy metal sequestration. The tested gene here was pcoA from Escherichia coli (Brown et al., 1995), which was also cloned in pBI121 and introduced in Nicotiana tabacum through an Agrobacterium tumefaciens transformation in a way similar as described for cadA. Kanamycine resistant transformants were obtained after transformation. All the kanamycine resistant transformants tested showed an increased resistance to copper (tested by a leaf disk assay) compared to the wild type and transformant with the pBI121 vector without gene (FIG. 3).

[0044] The pcoA protein has many closely related members, found to be involved in resistance against Cu. In addition, other proteins of these copper resistance determinants have also been shown to be involved in Cu sequestration, such as PcoC/CopC and CopE. These proteins, although different in structure, are also active in the bacterial periplasm and possess similar heavy metal binding sites as pcoA. In addition, a CopE like protein, referred to as SilE, was identified in the Salmonella sil operon encoding for Ag-resistance. The potential genes whose heterologous expression can result in improved resistance, are summarised in table 2. Genes Sequence ID Metals References cop operon (copA, C) M19930 Cu Mellano and e.g. of Pseudomonas Cooksey syringae (1988) pco operon (pcoA, C) G619126 Cu Brown et al., of e.g. E. coli 1995 PcoE X83541 Cu Brown et al., 1995 sil operon of AF067954, Ag Gupta et al., Salmonella nucleotide 1999 sequence sil operon

[0045] Use of the Genetically Modified Plants or Plant Cells According to the Invention:

[0046] Plants or plant cells according to the present invention can be used in several applications. It is clear that a plant according to the invention can be used for phytoremediation of contaminated sites, for revegetation, phytostabilisation, phytoextraction of contaminated soils and/or water. While the genetic modification allow the plants to grow in such contaminated environments, they will accumulate trace elements and remove them from the soil and/or water.

[0047] Another possible application is to grow said genetically modified plants or plant cells on a medium (solid or fluid) containing certain trace elements necessary or beneficial for human and animal health.

[0048] It would then be possible to prepare food/feed supplements from these plants containing beneficial trace elements which can be readily adsorbed. Further, the plants could serve as a basis for the preparation of a medicament.

REFERENCES

[0049] Silver S. et al. (1993). Molecular Microbiology 10(1): 7-12.

[0050] Horsch R. B. et al. (1985). Science 227: 1229-1231.

[0051] Karin M (1985). Metallothioneins: Proteins in search of function; Cell 41, 9-10.

[0052] Brown N. L. et al. (1995). Molecular Microbiology 17(6): 1153-1166.

[0053] Mellano, M. A. et al. (1988). J. Bacteriol. 170: 2879-2883.

[0054] Vulpe C. D., et al. (1993). Nature genet. 3: 7-13.

[0055] Petrukhin, K. et al. (1993). Nature Genet. 5 (4): 338-343.

[0056] Rugh C. L. et al. (1996). Proc. Natl. Acad. Sci. USA 93: 3182-3187.

[0057] Lefebvre, D. D. et al, 1987. Bio/Technology 5, 1053-1056.

[0058] Maiti, I. B. et al, 1988. Biochemical and Biophysical Research Communications 150, 640-647.

[0059] Maiti, I. B. et al, 1989. Plant Physiology 91, 1020-1024.

[0060] Maiti, I. B. et al. 1991. Plant Science 76, 99-107.

[0061] Misra, S. et al. 1989. Theoretical and Applied Genetics 78, 161-168.

[0062] Evans, K. M. et al 1992. Implications for PsMTA function. Plant Molecular Biology 20, 1019-1028.

[0063] Yeargan, R. et al 1992. Transgenic Research 1, 261-267.

[0064] Nucifora G. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86 (10): 3544-3548.

[0065] Rensing C., et al. (1998). J. Biol. Chem. 273: 32614-32617.

[0066] Rensing C., et al. (1997). Proc. Natl. Acad. Sci. U.S.A. 94 (26): 14326-14331.

[0067] Gupta, A., et al. 1999. Nature Medicine 5: 183-188.

[0068]

1 2 1 2217 DNA Artificial Sequence Staphylococcus aureus cadA gene with BamH I and Xba I restriction sites and plant specific translation signals 1 tctagattta ccacc atg tct gaa caa aag gtt aaa cta atg gaa gaa gaa 51 Met Ser Glu Gln Lys Val Lys Leu Met Glu Glu Glu 1 5 10 atg aac gtc tat cgg gtc caa gga ttt aca tgt gca aat tgt gca gga 99 Met Asn Val Tyr Arg Val Gln Gly Phe Thr Cys Ala Asn Cys Ala Gly 15 20 25 aag ttt gag aaa aat gtt aaa aag att cca ggc gtt cag gac gca aaa 147 Lys Phe Glu Lys Asn Val Lys Lys Ile Pro Gly Val Gln Asp Ala Lys 30 35 40 gta aac ttt ggc gct tct aaa att gat gta tat gga aat gca tcg gtt 195 Val Asn Phe Gly Ala Ser Lys Ile Asp Val Tyr Gly Asn Ala Ser Val 45 50 55 60 gaa gaa ctt gaa aaa gca ggt gct ttt gag aat cta aaa gta tct cct 243 Glu Glu Leu Glu Lys Ala Gly Ala Phe Glu Asn Leu Lys Val Ser Pro 65 70 75 gaa aaa cta gcg aat caa acg ata caa agg gtt aaa gat gac act aag 291 Glu Lys Leu Ala Asn Gln Thr Ile Gln Arg Val Lys Asp Asp Thr Lys 80 85 90 gct cat aaa gaa gag aaa aca cca ttt tat aaa aaa cat agt aca ttg 339 Ala His Lys Glu Glu Lys Thr Pro Phe Tyr Lys Lys His Ser Thr Leu 95 100 105 ctg ttt gcc aca cta cta att gct ttt ggt tac ctt tct cac ttt gta 387 Leu Phe Ala Thr Leu Leu Ile Ala Phe Gly Tyr Leu Ser His Phe Val 110 115 120 aat gga gaa gat aac ctc gta act tcc atg tta ttt gta ggt tct att 435 Asn Gly Glu Asp Asn Leu Val Thr Ser Met Leu Phe Val Gly Ser Ile 125 130 135 140 gta att ggc gga tat tca tta ttt aaa gtc ggt ttt caa aat ttg ata 483 Val Ile Gly Gly Tyr Ser Leu Phe Lys Val Gly Phe Gln Asn Leu Ile 145 150 155 cgc ttt gat ttc gac atg aaa acc ctg atg acc gtt gcc gtt att gga 531 Arg Phe Asp Phe Asp Met Lys Thr Leu Met Thr Val Ala Val Ile Gly 160 165 170 gct acc att att ggt aaa tgg gca gag gca tct att gtt gtt att ctc 579 Ala Thr Ile Ile Gly Lys Trp Ala Glu Ala Ser Ile Val Val Ile Leu 175 180 185 ttt gca atc agt gaa gca ctt gaa cgc ttc tct atg gac aga tca aga 627 Phe Ala Ile Ser Glu Ala Leu Glu Arg Phe Ser Met Asp Arg Ser Arg 190 195 200 caa tcc ata cgt tca ttg atg gat atc gcc cca aaa gaa gca cta gtt 675 Gln Ser Ile Arg Ser Leu Met Asp Ile Ala Pro Lys Glu Ala Leu Val 205 210 215 220 aga cga aat ggt cag gaa ata ata atc cat gtg gac gat atc gct gtg 723 Arg Arg Asn Gly Gln Glu Ile Ile Ile His Val Asp Asp Ile Ala Val 225 230 235 ggt gat atc atg att gtc aaa cca ggg gag aaa att gcc atg gat gga 771 Gly Asp Ile Met Ile Val Lys Pro Gly Glu Lys Ile Ala Met Asp Gly 240 245 250 atc att gtg aat ggc ttg tcg gct gtc aat cag gca gct ata aca gga 819 Ile Ile Val Asn Gly Leu Ser Ala Val Asn Gln Ala Ala Ile Thr Gly 255 260 265 gaa tct gtt ccc gtc tcc aaa gcg gta gat gac gaa gta ttt gca ggt 867 Glu Ser Val Pro Val Ser Lys Ala Val Asp Asp Glu Val Phe Ala Gly 270 275 280 acg ctt aac gaa gag gga cta att gaa gta aaa atc acc aaa tac gta 915 Thr Leu Asn Glu Glu Gly Leu Ile Glu Val Lys Ile Thr Lys Tyr Val 285 290 295 300 gaa gat aca acc att acc aag att att cat ctt gtt gaa gaa gca caa 963 Glu Asp Thr Thr Ile Thr Lys Ile Ile His Leu Val Glu Glu Ala Gln 305 310 315 ggg gag cgt gct cca gcc caa gca ttc gtt gat aaa ttt gcg aaa tac 1011 Gly Glu Arg Ala Pro Ala Gln Ala Phe Val Asp Lys Phe Ala Lys Tyr 320 325 330 tac act ccg atc att atg gtt att gca gcc ttg gtt gca gtc gtt cca 1059 Tyr Thr Pro Ile Ile Met Val Ile Ala Ala Leu Val Ala Val Val Pro 335 340 345 ccc cta ttc ttt ggt ggc agt tgg gat aca tgg gtt tat caa gga tta 1107 Pro Leu Phe Phe Gly Gly Ser Trp Asp Thr Trp Val Tyr Gln Gly Leu 350 355 360 gca gtt ctt gta gtt gga tgt cct tgt gca tta gtt att tct act cca 1155 Ala Val Leu Val Val Gly Cys Pro Cys Ala Leu Val Ile Ser Thr Pro 365 370 375 380 atc tcg att gtc tcg gca att gga aat gca gcg aaa aaa ggt gtg ttg 1203 Ile Ser Ile Val Ser Ala Ile Gly Asn Ala Ala Lys Lys Gly Val Leu 385 390 395 gtt aaa ggt ggt gtc tat ctc gag aaa tta gga gcc att aag aca gtc 1251 Val Lys Gly Gly Val Tyr Leu Glu Lys Leu Gly Ala Ile Lys Thr Val 400 405 410 gca ttt gat aaa aca gga aca ctg aca aaa ggt gta cca gtg gta aca 1299 Ala Phe Asp Lys Thr Gly Thr Leu Thr Lys Gly Val Pro Val Val Thr 415 420 425 gat ttt gaa gta tta aat gac caa gtg gaa gaa aaa gag cta ttc tct 1347 Asp Phe Glu Val Leu Asn Asp Gln Val Glu Glu Lys Glu Leu Phe Ser 430 435 440 atc att aca gct tta gaa tat cgt tca caa cat cca ctt gct tca gca 1395 Ile Ile Thr Ala Leu Glu Tyr Arg Ser Gln His Pro Leu Ala Ser Ala 445 450 455 460 ata atg aaa aag gca gag caa gat aat atc cct tat tct aat gta caa 1443 Ile Met Lys Lys Ala Glu Gln Asp Asn Ile Pro Tyr Ser Asn Val Gln 465 470 475 gtg gaa gaa ttc act tcg att act ggg cga ggt ata aaa ggg att gta 1491 Val Glu Glu Phe Thr Ser Ile Thr Gly Arg Gly Ile Lys Gly Ile Val 480 485 490 aac gga act act tac tat att gga agc cca aaa ctt ttc aag gaa tta 1539 Asn Gly Thr Thr Tyr Tyr Ile Gly Ser Pro Lys Leu Phe Lys Glu Leu 495 500 505 aat gtt tcc gat ttt agc ctt ggg ttt gaa aac aat gtg aaa atc cta 1587 Asn Val Ser Asp Phe Ser Leu Gly Phe Glu Asn Asn Val Lys Ile Leu 510 515 520 caa aac caa gga aaa aca gcc atg att att gga acg gaa aaa aca att 1635 Gln Asn Gln Gly Lys Thr Ala Met Ile Ile Gly Thr Glu Lys Thr Ile 525 530 535 540 ctc ggc gta att gcc gtt gca gat gag gtt cgt gaa aca agt aaa aat 1683 Leu Gly Val Ile Ala Val Ala Asp Glu Val Arg Glu Thr Ser Lys Asn 545 550 555 gtg att caa aaa ctt cat cag tta ggt atc aag caa aca att atg ctg 1731 Val Ile Gln Lys Leu His Gln Leu Gly Ile Lys Gln Thr Ile Met Leu 560 565 570 aca ggt gat aat caa ggt act gca aat gca atc ggt aca cat gta ggc 1779 Thr Gly Asp Asn Gln Gly Thr Ala Asn Ala Ile Gly Thr His Val Gly 575 580 585 gtt tct gat att cag tct gaa ttg atg cca cag gat aaa tta gat tat 1827 Val Ser Asp Ile Gln Ser Glu Leu Met Pro Gln Asp Lys Leu Asp Tyr 590 595 600 att aaa aaa atg caa tcg gag tat gat aat gta gct atg att ggc gat 1875 Ile Lys Lys Met Gln Ser Glu Tyr Asp Asn Val Ala Met Ile Gly Asp 605 610 615 620 ggc gtt aat gat gct cca gca ctt gct gca tct act gtt gga att gca 1923 Gly Val Asn Asp Ala Pro Ala Leu Ala Ala Ser Thr Val Gly Ile Ala 625 630 635 atg ggc ggt gct gga acg gat act gca att gaa aca gct gat att gca 1971 Met Gly Gly Ala Gly Thr Asp Thr Ala Ile Glu Thr Ala Asp Ile Ala 640 645 650 tta atg gga gat gat tta agt aag ctt cca ttt gca gta aga ctc agt 2019 Leu Met Gly Asp Asp Leu Ser Lys Leu Pro Phe Ala Val Arg Leu Ser 655 660 665 cga aaa act tta aat atc att aaa gct aac atc act ttt gct atc gga 2067 Arg Lys Thr Leu Asn Ile Ile Lys Ala Asn Ile Thr Phe Ala Ile Gly 670 675 680 att aaa ata att gcc tta cta tta gtt atc ccg gga tgg tta acc ctt 2115 Ile Lys Ile Ile Ala Leu Leu Leu Val Ile Pro Gly Trp Leu Thr Leu 685 690 695 700 tgg ata gcg att ctt tcc gat atg gga gct act att ttg gta gca tta 2163 Trp Ile Ala Ile Leu Ser Asp Met Gly Ala Thr Ile Leu Val Ala Leu 705 710 715 aat agt tta cga ctg atg aga gtg aag gat aaa taggtaatga tgtttggatc 2216 Asn Ser Leu Arg Leu Met Arg Val Lys Asp Lys 720 725 c 2217 2 727 PRT Artificial Sequence Staphylococcus aureus cadA gene with BamH I and Xba I restriction sites and plant specific translation signals 2 Met Ser Glu Gln Lys Val Lys Leu Met Glu Glu Glu Met Asn Val Tyr 1 5 10 15 Arg Val Gln Gly Phe Thr Cys Ala Asn Cys Ala Gly Lys Phe Glu Lys 20 25 30 Asn Val Lys Lys Ile Pro Gly Val Gln Asp Ala Lys Val Asn Phe Gly 35 40 45 Ala Ser Lys Ile Asp Val Tyr Gly Asn Ala Ser Val Glu Glu Leu Glu 50 55 60 Lys Ala Gly Ala Phe Glu Asn Leu Lys Val Ser Pro Glu Lys Leu Ala 65 70 75 80 Asn Gln Thr Ile Gln Arg Val Lys Asp Asp Thr Lys Ala His Lys Glu 85 90 95 Glu Lys Thr Pro Phe Tyr Lys Lys His Ser Thr Leu Leu Phe Ala Thr 100 105 110 Leu Leu Ile Ala Phe Gly Tyr Leu Ser His Phe Val Asn Gly Glu Asp 115 120 125 Asn Leu Val Thr Ser Met Leu Phe Val Gly Ser Ile Val Ile Gly Gly 130 135 140 Tyr Ser Leu Phe Lys Val Gly Phe Gln Asn Leu Ile Arg Phe Asp Phe 145 150 155 160 Asp Met Lys Thr Leu Met Thr Val Ala Val Ile Gly Ala Thr Ile Ile 165 170 175 Gly Lys Trp Ala Glu Ala Ser Ile Val Val Ile Leu Phe Ala Ile Ser 180 185 190 Glu Ala Leu Glu Arg Phe Ser Met Asp Arg Ser Arg Gln Ser Ile Arg 195 200 205 Ser Leu Met Asp Ile Ala Pro Lys Glu Ala Leu Val Arg Arg Asn Gly 210 215 220 Gln Glu Ile Ile Ile His Val Asp Asp Ile Ala Val Gly Asp Ile Met 225 230 235 240 Ile Val Lys Pro Gly Glu Lys Ile Ala Met Asp Gly Ile Ile Val Asn 245 250 255 Gly Leu Ser Ala Val Asn Gln Ala Ala Ile Thr Gly Glu Ser Val Pro 260 265 270 Val Ser Lys Ala Val Asp Asp Glu Val Phe Ala Gly Thr Leu Asn Glu 275 280 285 Glu Gly Leu Ile Glu Val Lys Ile Thr Lys Tyr Val Glu Asp Thr Thr 290 295 300 Ile Thr Lys Ile Ile His Leu Val Glu Glu Ala Gln Gly Glu Arg Ala 305 310 315 320 Pro Ala Gln Ala Phe Val Asp Lys Phe Ala Lys Tyr Tyr Thr Pro Ile 325 330 335 Ile Met Val Ile Ala Ala Leu Val Ala Val Val Pro Pro Leu Phe Phe 340 345 350 Gly Gly Ser Trp Asp Thr Trp Val Tyr Gln Gly Leu Ala Val Leu Val 355 360 365 Val Gly Cys Pro Cys Ala Leu Val Ile Ser Thr Pro Ile Ser Ile Val 370 375 380 Ser Ala Ile Gly Asn Ala Ala Lys Lys Gly Val Leu Val Lys Gly Gly 385 390 395 400 Val Tyr Leu Glu Lys Leu Gly Ala Ile Lys Thr Val Ala Phe Asp Lys 405 410 415 Thr Gly Thr Leu Thr Lys Gly Val Pro Val Val Thr Asp Phe Glu Val 420 425 430 Leu Asn Asp Gln Val Glu Glu Lys Glu Leu Phe Ser Ile Ile Thr Ala 435 440 445 Leu Glu Tyr Arg Ser Gln His Pro Leu Ala Ser Ala Ile Met Lys Lys 450 455 460 Ala Glu Gln Asp Asn Ile Pro Tyr Ser Asn Val Gln Val Glu Glu Phe 465 470 475 480 Thr Ser Ile Thr Gly Arg Gly Ile Lys Gly Ile Val Asn Gly Thr Thr 485 490 495 Tyr Tyr Ile Gly Ser Pro Lys Leu Phe Lys Glu Leu Asn Val Ser Asp 500 505 510 Phe Ser Leu Gly Phe Glu Asn Asn Val Lys Ile Leu Gln Asn Gln Gly 515 520 525 Lys Thr Ala Met Ile Ile Gly Thr Glu Lys Thr Ile Leu Gly Val Ile 530 535 540 Ala Val Ala Asp Glu Val Arg Glu Thr Ser Lys Asn Val Ile Gln Lys 545 550 555 560 Leu His Gln Leu Gly Ile Lys Gln Thr Ile Met Leu Thr Gly Asp Asn 565 570 575 Gln Gly Thr Ala Asn Ala Ile Gly Thr His Val Gly Val Ser Asp Ile 580 585 590 Gln Ser Glu Leu Met Pro Gln Asp Lys Leu Asp Tyr Ile Lys Lys Met 595 600 605 Gln Ser Glu Tyr Asp Asn Val Ala Met Ile Gly Asp Gly Val Asn Asp 610 615 620 Ala Pro Ala Leu Ala Ala Ser Thr Val Gly Ile Ala Met Gly Gly Ala 625 630 635 640 Gly Thr Asp Thr Ala Ile Glu Thr Ala Asp Ile Ala Leu Met Gly Asp 645 650 655 Asp Leu Ser Lys Leu Pro Phe Ala Val Arg Leu Ser Arg Lys Thr Leu 660 665 670 Asn Ile Ile Lys Ala Asn Ile Thr Phe Ala Ile Gly Ile Lys Ile Ile 675 680 685 Ala Leu Leu Leu Val Ile Pro Gly Trp Leu Thr Leu Trp Ile Ala Ile 690 695 700 Leu Ser Asp Met Gly Ala Thr Ile Leu Val Ala Leu Asn Ser Leu Arg 705 710 715 720 Leu Met Arg Val Lys Asp Lys 725 

1. Genetically modified plant or plant cell having an increased heavy metal resistance and comprising at least one nucleotide sequence encoding a protein selected from the group consisting of heterologous heavy metal transporter or sequestration proteins.
 2. The genetically modified plant or plant cell as in claim 1, characterised in that said nucleotide sequence is a prokaryotic nucleotide sequence.
 3. The genetically modified plant or plant cell as in claim 1 or 2, wherein the heavy metal transporter is a transporter selected from the group consisting of P-type ATPases, 3 components efflux pumps, ABC transporters or Cation Diffusion Facilitor proteins.
 4. The genetically modified plant or plant cell according to the claim 3, wherein the transporter is a cadmium ATPase.
 5. The genetically modified plant or plant cell according to the claim 3 or 4, wherein the nucleotide sequence is cadA or a portion thereof allowing heavy metal transport.
 6. The genetically modified plant or plant cell according to the claim 1, wherein the heavy metal sequestration protein belongs to the copA family.
 7. Method for inducing or increasing heavy metal resistance into a plant or plant cell, comprising the steps of: preparing a nucleotide sequence encoding a protein selected from the group consisting of heterologous heavy metal transporters or sequestration proteins, being operably linked to one or more regulatory sequences active into a plant, transforming a plant or plant cell with said nucleotide sequence, and possibly regenerating a plant from the transformed plant cell.
 8. Use of the genetically modified plant or plant cell according to any of the preceding claims 1 to 6 for the phytoremediation of contaminated heavy metals.
 9. The use according to the claim 8, wherein the phytoremediation of contaminated sites is selected from the group consisting of revegetation, phytostabilisation, phytoextraction of soils and/or water contaminated with trace elements.
 10. Pharmaceutical composition comprising an adequate pharmaceutical carrier and a sufficient amount of the genetically modified plant or plant cell according to any of the preceding claims 1 to
 6. 11. Feed or food composition or additive comprising the genetically modified plant or plant cell according to any of the preceding claims. 